Functional and structural characterization of DR_0079 from Deinococcus radiodurans, a novel Nudix hydrolase with a preference for cytosine (deoxy) ribonucleoside 5'-di- and triphosphates
The Deinococcus radiodurans Nudix hydrolase DR0079 was assayed for activity towards a wide variety of substrates and observed to have a marked specificity for cytosine ribonucleoside 5’-diphosphate (CDP) and cytosine ribonucleoside 5’-triphosphate (CTP) with a slight preference for CDP. The next most specific substrates, with a relative activity of <50%, were the corresponding deoxyribose nucleosides, dCDP and dCTP. Enzyme hydrolase activity at the site of the phosphodiester bond was corroborated using 31P NMR spectroscopy to follow the phosphorus resonances for two substrates, CDP and IDP, and the hydrolysis products, NMP and Pi. Optimum activity for CDP was determined to be at pH 9.0 – 9.5. The optimal divalent cation for CDP hydrolysis at this pH was Mg2+ followed by Mn2+ (~47%) and Co2+(~27%). The biochemical data is discussed with reference to the crystal structure for the D. radiodurans DR0079 that was determined in the apo-metal form at 1.9 Å resolution. The protein in the crystal structure contains nine β-strands, three α-helices, and two 3-10 helices that are organized into three subdomains; an N-terminal β-sheet, a central Nudix core, and a C-terminal helixturn- helix motif. As observed for all known structures of Nudix hydrolases, the α-helix of the ‘Nudix box’ is one of two helices that sandwich a ‘four-strand’ mixed β-sheet. Using 15N-labelled DR0079, NMR chemical shift mapping experiments were performed with the paramagnetic divalent cation Co2+ and the non-hydrolyzable substrate thymidine- 5’-O-(α,β-methylenediphosphate (TMP-CP). The results of the chemical shift perturbation experiments were mapped onto the crystal structure of DR0079 and a model for substrate binding proposed.
- Research Organization:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (US), Environmental Molecular Sciences Laboratory (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 935898
- Report Number(s):
- PNNL-SA-58409; 2331a; 3332; KP1504020
- Journal Information:
- Biochemistry, 47(25):6571-82, Journal Name: Biochemistry, 47(25):6571-82 Journal Issue: 25 Vol. 47
- Country of Publication:
- United States
- Language:
- English
Similar Records
Solution structure of hypothetical nudix hydrolase dr0079 from extremely radiation-resistant deinococcus radiodurans bacterium
Solution Structure of Hypothetical Nudix Hydrolase DR0079 from Extremely Radiation-Resistant Deinococcus radiodurans Bacterium
Related Subjects
CATIONS
CHEMICAL SHIFT
CRYSTAL STRUCTURE
CYTOSINE
DEOXYRIBOSE
ENZYMES
Environmental Molecular Sciences Laboratory
FUNCTIONALS
HYDROLASES
HYDROLYSIS
NUCLEOSIDES
PHOSPHORUS
PROTEINS
RESOLUTION
SPECIFICITY
SPECTROSCOPY
SUBSTRATES
chemical shift mapping
desiccation
function screen
phosphorus-31 NMR
polyphosphate pyrophosphohydrolases
pseudo contact shifts